Chemical and method for analyzing DNA sequence by visualizing single-molecule DNA and use thereof

ABSTRACT

The present disclosure relates to a composition for analysis of DNA sequences and a method for analysis of DNA sequences by using the same and, more particularly, to a composition comprising the compound represented by Chemical Formula 1 and a method for analysis of DNA sequences, the method comprising a step of treating a sample with the same. The compound represented by Chemical Formula 1 in which TAMRA is linked to polypyrrole specifically binds an A/T base pair (W) to fluoresce alone without causing DNA photocleavage. Therefore, the compound is useful for DNA analysis particularly at the single DNA molecule level.

TECHNICAL FIELD

The present disclosure relates to a composition for analysis of DNAsequences and a method for analysis of DNA sequences by using the sameand, more particularly, to a composition comprising the compoundrepresented by Chemical Formula 1 and a method for analysis of DNAsequences, the method comprising a step of treating a sample with thesame.

The present patent application claims priority to and the benefit ofKorean Patent Application No. 10-2018-0086255 filed in the KoreanIntellectual Property Office on Jul. 24, 2018, the entire contents ofwhich are incorporated herein by reference.

BACKGROUND ART

Direct visualization of individual DNA molecules is very importantbecause it allows for understanding biochemical events within thecontext of DNA sequences. Although sequencing technology at the singlenucleotide level has advanced, biological problems still remainunsolved, which are limited by short read length and information losswithin a large genome.

The ultimate goal of DNA analysis would be to acquire nucleotidesequences and epigenetic information directly from chromosomal DNAwithout fragmentation or amplification. Given these concerns, single DNAmolecules are a promising platform to overcome limitations of currentsequencing technology.

In this regard, optical mapping, which is a technique for gaininggenetic information by visualizing a large DNA molecule, has beencontinually developed. This technique is a method to make barcode-likepatterns from a single DNA molecule for visualization.

Meanwhile, conventional analysis methods using sequence-specificrestriction enzymes retain the fundamental problem of DNA cleavage.Analysis methods using sequence-specific substances for A/T base pairs(Netropsin, etc.) and fluorescent dye markers raises the problem thatYOYO-1, used as the fluorescent dye, causes light-induced DNA cleavage.

There is therefore a need for the development of a substance that canbind in a sequence-specific manner and fluoresce alone without causingDNA cleavage.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

The present inventors endeavored to develop a composition capable ofbinding in a sequence-specific manner without DNA cleavage, and as aresult, the present inventors confirmed that TAMRA-linked polypyrrolecan bind specifically to A/T base pairs (W) and fluoresce alone withoutDNA photocleavage, and thus the present inventors completed the presentinvention.

Therefore, a purpose of the present disclosure is to provide acomposition for analysis of DNA sequences, the composition comprising acompound represented by Chemical Formula 1.

Another purpose of the present discloses is to provide a method foranalysis of DNA sequences, the method comprising a step of applying aDNA sequencing composition comprising a compound represented by ChemicalFormula 1.

Technical Solution

The present inventors endeavored to develop a composition capable ofbinding in a sequence-specific manner without DNA cleavage, and as aresult, the present inventors confirmed that TAMRA-linked polypyrrolecan bind specifically to A/T base pairs (W) and fluoresce alone withoutDNA photocleavage.

The present disclosure pertains to a composition for analysis of DNAsequences, the composition comprising a compound represented by ChemicalFormula 1 and a method for analysis of DNA sequences, the methodcomprising a step of treating a sample therewith.

Below, a detailed description will be given of the present disclosure.

In accordance with an aspect of the present invention, there is provideda composition for analysis of DNA sequences, the composition comprisinga compound represented by the following Chemical Formula 1:

wherein,

n, m, o, and p are each independently an integer of 1 to 10, and

X may be a fluorescent protein, a photoprotein, a colorreaction-catalyst, biotin, a fluorescent substance, a luminescentsubstance, or a chemiluminescent substance.

The color reaction-catalyst may be, but not limited to, alkalinephosphatase, peroxidase, β-galactosidase, and/or β-glucosidase.

The fluorescent substance may be, but not limited to, TAMRA(carboxytetramethylrhodamine), fluorescein), Cy5 (Cyanine 5), Cy3(Cyanine 3), HEX (5′-Hexachloro-Fluorescein), TET(5′-Tetrachloro-Fluorescein), Dabsyl (4-(dimethylaminoazo)benzene-4-carboxylic acid), and/or FAM (Fluorescein amidite).

In Chemical Formula 1, n, m, o, and p may each be independently aninteger of 1 to 5.

In Chemical Formula 1, X may be as follows:

In addition, the compound represented by Chemical Formula 1 may be acompound represented by the following Chemical Formula 2:

The composition may bind specifically to an adenine/thymine (A/T) basepair (W).

The DNA may be, but not limited to, a single DNA molecule, a chromosome,or a chromatin fiber.

The composition binds DNA via a hydrogen bonding interaction between apolypyrrole and the minor-groove of DNA while the TAMRA(carboxytetramethylrhodamine) moiety remains far from the DNA backbone.Thus, the composition has the advantage of suppressing the DNAphotocleavage, which is a significant problem with the conventional DNAdye YOYO-1 and thus does not cleave DNA during repeated cycles of DNAelongation and relaxation.

Moreover, the composition can analyze chemically modified or damaged DNAsequences or backbones, unlike typical sequencing approaches, and canbe, thus, effectively used at the single DNA molecule level.

In accordance with another aspect of the present invention, there isprovided a method for analysis of DNA sequences comprising: a step oftreating a sample with a compound represented by the Chemical Formula 1:

wherein,

n, m, o, and p are each independently an integer of 1 to 10,

X may be a fluorescent protein, a photoprotein, a colorreaction-catalyst, biotin, a fluorescent substance, a luminescentsubstance, or a chemiluminescent substance.

The method may further comprise: a step of comparing an entire genomicadenine/thymine (A/T) frequency in a subject to be analyzed and an A/Tfrequency of the sample treated with the composition.

The composition may specifically bind to an adenine/thymine (A/T) basepair (W).

The sample may be, but not limited to, a genetic material comprising asingle DNA molecule, an oligo DNA, a chromosome, a polytene chromosome,or a chromatin fiber.

In the method, a target DNA sequence may be analyzed using, for example,a Python program in which the entire genomic A/T frequency of a subjectto be analyzed is scanned through in silico map and a search is made ofthe best alignment position between the image of the sample treated withthe composition and the scanned entire genomic A/T frequency.

The overlapping description of the composition is omitted inconsideration of the complexity of the specification.

Advantageous Effects

The present invention is directed a composition for analysis of DNAsequences and a method for analysis of DNA sequences by using the sameand, more particularly, to a composition comprising the compoundrepresented by Chemical Formula 1 and a method for analysis of DNAsequences, the method comprising a step of treating a sample with thesame.

The compound of the present disclosure, represented by Chemical Formula1, in which TAMRA is linked to a polypyrrole, specifically binds an A/Tbase pair (W) to fluoresce alone, without DNA photocleavage and can bethus useful particularly for DNA analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a DNA molecule stainedspecifically at adenine (A) and thymine (T) rich regions according to anembodiment of the present disclosure.

FIG. 2a is a view of λ DNA (48.5 kbp) molecules stained according to anembodiment of the present disclosure that have a mushroom-likeconformation in the flow off condition.

FIG. 2b is a view of λ DNA molecules stained according to an embodimentof the present invention that are fully elongated in a 100 μL/min flowcondition.

FIG. 2c is a view of false-labeled DNA molecules stained according to aComparative Example.

FIG. 3a is a view showing the comparison of experimentally measuredfluorescence intensity with in silico sequence frequencies for genomesequences (W, W₄, and W₉) from the λ genome sequence stained accordingto an embodiment of the present disclosure.

FIG. 3b is a view showing the comparison of cross-correlationcoefficient values calculated from the alignment of 20 molecular imageswith the λ DNA genome.

FIG. 4 shows photo-cleavage gel electrophoresis assay results of λ DNAstained with the compound according to an embodiment of the presentdisclosure and YOYO-1.

FIG. 5a shows images of tangled and spread polytene chromosomes from D.melanogaster after staining according to an embodiment of the presentdisclosure.

FIG. 5b shows fluorescent λ DNA images stained with the compound of anembodiment of the present disclosure and DAPI.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes only and are not to be construedto limit the scope of the present invention.

Preparation Example: Preparation of TAMRA-β₂-Py₄-β-Py₄-Dp

Fmoc-Py-COOH, an oxime resin, HCTU, DIEA, and dimethylformamide (DMF)were used to afford 50 mg of an Fmoc-Py-oxime resin (0.40 mmol/g).

Briefly, 20% piperidine/DMF (500 μL) was deblocked twice for 4 minutes,and 10% DIEA/N-methyl pyrrolidone (NMP) (364 μL, 0.21 mmol DIEA) wasadded to an Fmoc-Py-COOH (77 mg, 0.21 mmol) or Fmoc-β-COOH (66 mg, 0.21mmol)-HCTU mixture, followed by a coupling reaction for 60 minutes(sequentially, four times with Fmoc-Py-COOH, once with Fmoc-β-COOH, fourtimes with Fmoc-Py-COOH, and twice with Fmoc-β-COOH). The reactionmixture was washed five times with DMF after each step. After the lastcoupling reaction, the amino group at the N-terminus was deprotected.All coupling reactions were carried out with a single-coupling cycle.All lines were purged with solution and bubbled by nitrogen gas forstirring the resin.

Subsequently, polypyrrole on the resin was cleaved with 0.60-1.0 mL ofN, N-dimethyl-1,3-propanediamine or 3,3′-diamino-N-methyldipropylamineat 55° C. for 3 hours. The resin was removed by filtration and washedthoroughly with dichloromethane, and the filtrate was concentrated in avacuum. The residue was dissolved in 1.0 to 2.0 mL of adichloromethane-methanol mixture and then more than 10-fold volumes ofdiethyl ether was added, followed by centrifugation at 10,000×g for 10minutes and removal of the supernatant. This process was repeated untilwhile precipitates were obtained.

The crude product thus obtained was purified by reversed-phase flashchromatography (CombiFlash Rf, Teledyne Isco, Lincoln, Nebr.) using a4.3 g reversed-phase flash column (C18 RediSep Rf) in 0.1% TFA (inwater) with acetonitrile serving as an eluent. Collected fractions werelyophilized to obtainH₂N-β-alanine)₂-(N-methylpyrrole)₄-(β-alanine)-(N-methylpyrrole)₄-(3-(dimethylamino)propylamine)(H₂N-β₂-Py₄-β-Py₄-Dp) (19 mg, 1.5×10⁻² mmol).

Afterward, H₂N-β₂-Py₄-β-Py₄-Dp (1.3 mg, 1.0×10⁻³ mmol) obtained aboveand 5-TAMRA NHS ester (1.2 mg, 2.3×10⁻³ mmol) were dissolved in DMF (190μL) and DIEA (0.70 μL, 4.0×10⁻³ mmol) and then mixed at roomtemperature, with light shielded therefrom. The reaction mixture waspurified by reversed-phase high performance liquid chromatography(HPLC), followed by lyophilization to afford(5-carboxytetramethylrhodamine)-(β-alanine)₂-(N-methylpyrrole)₄-(β-alanine)-(N-methylpyrrole)₄-(3-(dimethylamino)propylamine)(TAMRA-β₂-Py₄-β-Py₄-Dp) as a purple powder (1.7 mg, 1.0×10⁻³ mmol,quant).

HPLC: t_(R)=18.2 min. MALDI-TOF-MS m/z calcd for C₈₇H₉₈N₂₃O₁₅ ⁺[M+H]⁺1704.761 found 1704.766.

Comparative Example 1: Preparation of TAMRA-β₂-Py₄-Dp

H₂N-β₂-Py₄-Dp was prepared in the same manner as in the PreparationExample, with the exception of using 100 mg of an Fmoc-Py-oxime resin(0.36 mmol/g) according to the different number of the couplingreactions of Fmoc-Py-COOH or Fmoc-β-COOH (sequentially, twice withFmoc-β-COOH and four times with Fmoc-Py-COOH).

Afterwards, H₂N-β₂-Py₄-Dp (1.1 mg, 1.5×10⁻³ mmol) was dissolved in DMF(140 μL) and DIEA (0.52 μL, 3.0×10⁻³ mmol) and mixed at roomtemperature, with light shielded therefrom. The reaction mixture waspurified by reversed-phase high-performance liquid chromatography,followed by lyophilization to afford(5-carboxytetramethylrhodamine)-(β-alanine)₂-(N-methylpyrrole)₄-(3-(dimethylamino)propylamine)(TAMRA-β₂-Py₄-Dp)as a purple powder (1.5 mg, 1.3×10⁻³ mmol).

HPLC: t_(R)=17.2 min. MALDI-TOF-MS m/z calcd for C₆₀H₆₉N₁₄O₁₀ ⁺[M+H]⁺1145.532 found 1145.559.

Comparative Example 2: Preparation of TAMRA-β₂-Py₄-TAMRA

H₂N-β₂-Py₄-NH₂ was prepared in the same manner as in the PreparationExample, with the exception of using 85 mg of an Fmoc-Py-oxime resin(0.27 mmol/g) according to the different number of the couplingreactions of Fmoc-Py-COOH or Fmoc-β-COOH (sequentially, twice withFmoc-β-COOH and four times with Fmoc-Py-COOH).

Afterward, H₂N-β₂-Py₄-NH₂ (2.3 mg, 3.0×10⁻³ mmol) and 5-TAMRA NHS ester(3.1 mg, 5.9×10⁻³ mmol) were dissolved in DMF (200 μL) and DIEA (10 μL,5.7×10⁻² mmol) and mixed at room temperature, with light shieldedtherefrom. The reaction mixture was purified by reversed-phasehigh-performance liquid chromatography, followed by lyophilization toafford(5-carboxytetramethylrhodamine)-(β-alanine)₂-(N-methylpyrrole)₄-(5-carboxytetramethylrhodamine)(TAMRA-β₂-Py₄-TAMRA)as a purple powder (1.2 mg, 7.5×10⁻⁴ mmol).

HPLC: t_(R)=18.2 min. MALDI-TOF-MS m/z calcd for C₈₇H₉₄N₁₇O₁₄ ⁺[M+H]⁺1600.716 found 1600.779.

Fluorescence Microscopy and DNA Visualization

An inverted microscope (Olympus IX70, Tokyo, Japan) with 60× OlympusUPlanSApo oil immersion objectives illuminated with an LED light source(SOLA SM II light engine, Lumencor, Beaverton, Oreg., USA) was used. Thelight was passed through corresponding filter sets (Semrock, Rochester,N.Y., USA) to set the excitation and emission wavelengths. A maximumlight intensity of 140 mW/cm² was measured. Fluorescence microscopicimages were taken with an electron-multiplying charge-coupled devicedigital camera (Evolve EMCCD, Roper Scientific, Tucson, Ariz., USA) andstored in a 16-bit TIFF format using the software Micro-manager. Forimage processing and analysis, ImageJ software with Java plug-ins andpython programs developed by the present inventors were utilized.

Python Program

-   -   ImageCompare.py: a library of functions,    -   seq2map.py: to convert a FASTA file into a A/T frequency in        silico map wherein a selected sequence is represented in white        for high frequency regions and in black for low frequency        regions.    -   insilicoMapFolder.py: to scan and compare in silico image files        and DNA images obtained through experiments with respect to all        images in the folder, to search positions of sites having the        highest cross-correlation coefficient, to convert the values,        and to store the values in new record files.    -   sortView.py: to read record files obtained with the        insilicoMapFolder.py, to compare signals, to search        cross-correlation coefficients, and to visualize image        comparison in a new window.    -   randomtiff.py: to generate random intensity tiff images

Experimental Example 1: Identification of DNA Staining at SingleMolecule Level

First, λ DNA (NCBI: NC_001416.1) was diluted to a concentration of 5ng/μL (0.16 nM; base pair 7.76 μM) in 1×TE (10 mM Tris, 1 mM EDTA, pH8.0) and mixed at a volume ratio of 1:1 with 70 μM of the PreparationExample solution. Next, the mixture was incubated at room temperaturefor 15 min and 20-fold diluted with 4% 3-mercaptoethanol (β-ME) in 1×TE.

Separately, a flow chamber (5×10×0.1 mm (L×W×H)) was prepared by placingan acrylic support on an acid-cleaned cover slip, with the walls formedby double-sided tape, An NE-1000 syringe pump (New Era Pump SystemsInc., Wantagh, N.Y.) was used to control the buffer.

Thereafter, 40 μg/mL biotinylated bovine serum albumin (BSA) wasinjected and incubated at room temperature for 10 min, after which adilution of 20 μg/mL Neutravidin in T50 solution (10 mM Tris, 50 nMNaCl, pH 8.0) was injected to the flow chamber and incubated at roomtemperature for 10 min.

Then, 1 μM of λ DNA overhang oligo(5′-p-GGGCGGCGACCT-Triethyleneglycol-biotin-3′) was loaded into the flowchamber and maintained at room temperature for 10 minutes. λ DNA, 200 Uof T4 DNA ligase, and reaction buffer were added and incubated at roomtemperature for 30 minutes.

After the residual enzyme mixture was washed with 1×TE, the dilutedPreparation Example solution was flowed into the channels, resulting invisualization of the tethered DNA. Stained DNA molecules were visualizedunder a continuous flow of 1×TE (100 μl/minute).

As shown in FIGS. 2a to 2c , the compound of the Preparation Examplevisualized λ DNA molecules with sequence specificity as well asenhancing the intensity of the molecules. The λ DNA molecules stainedwith the compound of the Preparation Example were free-floating with amushroom-like conformation (FIG. 2a ) and were fully elongated at a flowof 100 μL/minute (FIG. 2b ).

In contrast, the compound of Comparative Example 1 stained only in partAT-rich regions and bound undesired regions. The images were not brightenough to efficiently visualize the DNA backbone (FIG. 2c ). Inaddition, the compound of Comparative Example 2 did not binddouble-stranded DNA at all, resulting in no observation of fluorescence,probably because the steric hindrance of two TAMARAs at both endsinhibited tetra-pyrrole (Py₄) binding to DNA.

Experimental Example 2: Identification of DNA Binding at Single MoleculeLevel

The tethering possibility of the compound of the Preparation Example tothree candidates (W, W₄, and W₉) as binding sequences was identified asfollows: 1) the compound binds nine consecutive A/T base pairs (W₉); 2)tetra-pyrrole (Py₄) binds four consecutive A/T base pairs and the otherworks as a linker (W₄); and 3) the compound randomly interacts with oneA/T (W) rather than consecutive sequences.

Cross-correlation (cc) coefficient values calculated by comparing thealignment of 20 molecular images of above three kinds of bindingsequences (W, W₄ and W₉) with the genome. The control cross-correlationcoefficient (hereinafter referred to as cc) was obtained by comparingthe 100 computer-generated random sequences with the in silico images(***P<0.0001 for random-sequences paired t-test).

$\mspace{20mu}{{{Cross}\text{-}{correlation}\mspace{14mu}{{coefficient}(r)}} = \frac{\sum\limits_{i = 1}^{n}{\left( {x_{i} - \overset{\sim}{x}} \right)\left( {y_{i} - \overset{\sim}{y}} \right)}}{\sqrt{\sum\limits_{i = 1}^{n}{\left( {x_{i} - \overset{\sim}{x}} \right)^{2}\sqrt{\sum\limits_{i = 1}^{n}\left( {y_{i} - \overset{\sim}{y}} \right)^{2}}}}}}$$\left( {}^{n}{{= {{number}\mspace{11mu}{of}\mspace{11mu}{sample}}},x_{i},{y_{i} = {{value}\mspace{11mu}{at}\mspace{11mu}{each}\mspace{11mu}{site}}},\mspace{11mu}\overset{\_}{x},{\overset{\_}{y} = {{sample}\mspace{11mu}{average}}}} \right)$

As can be seen in FIGS. 3a and 3b , comparison was made betweenexperimentally measured fluorescence intensity and the threefluorescence intensity profiles (FIG. 3a ), indicating that theexperimental measurement agrees better with simple A/T frequency than W₄and W₉. In addition, cc was calculated using the Python program, showingthe highest cc value for simple A/T frequency (FIG. 3b ).

Experimental Example 3: Identification of DNA Staining-Induced DNAPhotocleavage

First, 1 μL of a λ DNA solution (500 ng/μL) was added to a restrictionenzyme (HindIII) reaction solution to form a total reaction volume of 50μL and the reaction was allowed to progress at 37° C. for 1 hour,followed by incubating at 65° C. for 15 minutes to inactivate therestriction enzyme digestion.

-   -   YOYO-1 stained DNA: YOYO-1 was mixed with 100 ng of the λ DNA to        form a final concentration of 4 μM, followed by incubation at        room temperature for 15 minutes. The mixture was exposed to 488        nm light source at room temperature for 30 minutes.    -   DNA stained with the compound of the Preparation Example: 100 ng        of the λ DNA was mixed with the compound of the Preparation        Example at a final concentration of 100 μM, followed by        incubation at room temperature for 15 minutes. Exposure was made        to 580 nm light for 30 minutes.    -   Control: a λ DNA solution containing no dyes and an        unilluminated λ DNA solution were used.

Each of the solutions was electrophoresed for 30 minutes on 0.7% agarosegel and observed.

As can be seen in FIG. 4, YOYO-1-stained DNA completely disappearedwhereas the DNA stained with the compound of the Preparation Example wasnot affected at all.

Experimental Example 4: Identification of DNA Staining at ChromosomalLevel

For comparison with DAPI, which is conventionally used to visualizepolytene chromosomes on a fluorescence microscope, the polytenechromosomes from the Drosophila melanogaster salivary gland were stainedwith the compound.

In brief, dissected salivary glands from third larvae of Drosophilamelanogaster were fixed with a solution containing a 1:2:3 ratio ofpropionic acid, deionized water, and acetic acid on a positively chargedcoverslip. Then, the cells were located between a slide glass and thecoverslip and then the individual cells were gently spread. After movingthe coverslip back and forth on the slide glass, the spread cells weresquashed for up to 15 minutes. Then, the slide glass and coverslip wereslightly dipped into liquid nitrogen. Just after no more bubbles weregenerated, the cover slip was removed from the slide glass. Finally, adilution of 2.5 μM of the compound of the Preparation Example in 4% β-MEwas used to stain the polytene chromosomal DNA.

FIG. 5a shows a typical image for tangled and spread polytenechromosomes, both of which clearly demonstrate band and interbandpatterns. In addition, the DNA stained with the compound of thePreparation Example DNA exhibits a clear backbone compared withDAPI-stained DNA, as shown in FIG. 5b . The compound of the PreparationExample is excited by yellow wavelengths (580) and does not damage DNA,whereas DAPI require ultraviolet light sources or equivalent lowwavelength light to excite the fluorophores, which can damage DNA.

CONCLUSION

Taken together, the data imply that the composition of the presentinvention specifically stains AT-rich regions in DNA and exhibitsdistinct fluorescence intensity patterns on DNA backbones when bindingDNA. Moreover, such a sequence-specific pattern allows the determinationof the DNA sequence from a microscopic image of a DNA fragment if giventhe full sequence. Therefore, the composition of the present inventioncan be effectively used for analyzing huge single DNA molecules at highspeed and high yield.

Staining polytene chromosomal DNA with the composition of the presentinvention can exhibit the band and interband patterns with ahigh-resolution, so that the composition of the present invention isuseful for studying somatic genome instability, chromosomal organizationof the genome, and protein immunolocalization.

What is claimed is:
 1. A composition for analysis of DNA sequences, thecomposition comprising a compound represented by the following ChemicalFormula 1: wherein, n is an integer of 2 to 10, m, o, and p are eachindependently an integer of 1 to 10, and X is one selected from thegroup consisting of a fluorescent protein, a photoprotein, a colorreaction-catalyst, biotin, a fluorescent substance, a luminescentsubstance, and a chemiluminescent substance.
 2. The composition of claim1, wherein the fluorescent substance is one selected from the groupconsisting of TAMRA (carboxytetramethylrhodamine), fluorescein, Cy5(Cyanine 5), Cy3 (Cyanine 3), HEX (5′-hexachloro-fluorescein), TET(5′-tetrachloro-fluorescein), Dabsyl(4-(dimethylaminoazo)benzene-4-carboxylic acid), and FAM (fluoresceinamidite).
 3. The composition of claim 1, wherein n is an integer of 2 to5; m, o, and p are each independently an integer of 1 to 5; and X is


4. The composition of claim 1, wherein the compound represented byChemical Formula 1 is a compound represented by the following ChemicalFormula 2:


5. The composition of claim 1, binding to an adenine/thymine (A/T) basepair (W).
 6. The composition of claim 1, wherein the DNA is one selectedfrom the group consisting of a single DNA molecule, a chromosome, and achromatin fiber.
 7. A method for analysis of DNA sequences comprising: astep of treating a sample with the composition of claim
 1. 8. The methodof claim 7, further comprising: a step of comparing an entire genomicadenine/thymine (A/T) frequency in a subject to be analyzed and an NTfrequency of the sample treated with the composition.
 9. The method ofclaim 7, wherein the composition binds to an A/T base pair (W).
 10. Themethod of claim 7, wherein the sample is at least one selected from thegroup consisting of a single DNA molecule, an oligo-DNA, a chromosome, apolytene chromosome, and a chromatin fiber.